FLAVINE NUCLEOTIDE NITRATE REDUCTASE FROM BROAD BEAN LEAVES

Hydrosulfite-reduced FMN served as an electron donor for nitrate reductase purified from broad bean leaves. FMN was successfully replaced with BV. The flavine nucleotide nitrate reductase had its pH optima at about 7.8 with phosphate buffer and at about 7.4 with Tris-HCl buffer. The Km's for nitrate and FMN were 3.7 × 10-4 M and 3.7 × 10-5 M, respectively.NADH2: nitrate reductase activity was completely inhibited by 0.1 mM p-CMB, whereas FMNH2: nitrate reductase activity was not. Inhibited activity was restored by the addition of cysteine. A sulfhydryl enzyme is involved in the NADH2: nitrate reductase system but not in the FMNH2 : nitrate reductase system. NADH2 and FMNH2 probably feed electrons into the electron transport chain at different sites. The nitrate reductase preparation had an NADH2-specific diaphorase activity which was almost completely inhibited by 0.1 mM p-CMB. The NADH2-specific diaphorase may form the sulfhydryl enzyme which mediates electron transfer between NADH2 and nitrate. © 1969 Oxford University Press.